The present invention is concerned with speciation of organisms, for the purpose of improving differential diagnosis of disease. The assays currently available to distinguish between or among species have not always met the expectations of consumers because they are either too costly, cumbersome or unavailable.
Polymerase chain reaction (PCR) and serological assays are currently used to distinguish among species. Serological tests present problems with cross-reactivity and available PCR tests are complicated. Typically, PCR-based assays require three steps: 1) conducting PCR using a primer set which distinguishes among members of different genera, but not among members of the same genus; 2) digesting the PCR products with restriction enzymes and 3) distinguishing among species on the basis of restriction digest patterns. One assay uses several sets of species-specific primers instead of digestion with restriction enzymes, with identification of the PCR products made by amplicon size. Minnick and Barbian, 31 J Microb Meth 51 (1997).
Haemobartonella felis, which causes infectious feline anemia, has two known subspecies: the California subspecies and the Ohio/Florida (herein called xe2x80x9cOhioxe2x80x9d) subspecies. Other organisms also cause anemia (eg. Bartonella and Ehrlichia), but treatment of the anemia is ideally directed to the causative organism. PCR technology has been used to detect Haemobartonella felis, although distinguishing between anemia-causing subspecies has not been accomplished.
In Rikihisa et al., 35 (4) J. Clin. Micros. 823 (1997), there is disclosed the use of portions of conserved 16S sequences as primers in order to sequence the 16S genes of H. felis California and H. felis Ohio, and evolutionarily compare them to each other as well as to other organisms. It also discloses a method for distinguishing H. felis strains from one another, which method requires restriction enzyme cleavage and gel electrophoresis. In Messick et al., 36 (2) J. Clin. Microb. 462 (1998), there are disclosed primers useful to identify (selectively) H. felis Ohio. It does not identify an assay for distinguishing H. felis Ohio and H. felis California or other organisms from H. felis Ohio.
In another organism, Bartonella PCR assays have been discussed which use differences in citrate synthase sequences. These assays use a first step of conducting PCR and a second step of digesting the PCR products with restriction enzymes to distinguish among species. Joblet et al., 33(7) J. Clin. Microb. 1879 (1995); Norman et al., 33(7) J. Clin. Microb. 1797 (1995). PCR assays on the basis of differences in 16S rRNA sequences in Bartonella have also been conducted, using restriction enzymes to distinguish among species. Birtles, 129 FEMS Microbiol. Letters 261 (1995).
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on subjective characterization of information available to the applicant, and does not constitute any admission as to the accuracy of the dates or contents of these documents.
The present invention requires only a single step to generate amplicons which identify a specific species.
It is therefore an object to provide a simplified assay for distinguishing between or among Haemobartonella species.
It is yet another object to provide materials related to the methods disclosed, including primer sets.
In all of the above embodiments, it is an object to provide methods to diagnose disease using the materials and methods provided.
It is also an object to provide methods for identifying primers useful to conduct PCR assays which capitalize on the species-specific size differences in the 16S region of Haemobartonella.
Finally, it is an object of the invention to provide a kit for convenient use of the materials and methods herein provided.
Definitions: For the purposes of the present invention, the following terms shall have the following definitions:
xe2x80x9cAmplicon(s)xe2x80x9d shall mean a nucleic acid produced through use of primers in PCR.
xe2x80x9cGenus-specific primer(s)xe2x80x9d shall mean primers being capable of amplifying an amplicon from at least a portion of the 16S region of at least two Haemobartonella species, and no other genera, and wherein the size of the amplicon is unique to the species.
When the term xe2x80x9cGenus-specific primer(s)xe2x80x9d is used to describe primers used in PCR assays, it is assumed that said primers are also being in amounts sufficient to amplify at least one ascertainable fragment.
A xe2x80x9csetxe2x80x9d of primers means at least one forward and at least one reverse primer, that when used in a PCR assay in appropriate amounts and in the presence of amplifiable nucleic acid, is capable of amplifying nucleic acid.
xe2x80x9cSpeciesxe2x80x9d means any species or subspecies, or other subset of species or subspecies.
In broad terms, the present invention includes materials and methods useful to distinguish between and among species of a genus. The methods simplify and are therefore more cost-effective than previous methods. In addition, because the present methods are simpler than previous methods, the risk of operator error, contamination, or any other technical problem is reduced, making the present invention inherently more reliable than previous methods.
The present invention also includes methods to detect Haemobartonella species in a test sample, comprising: a.) conducting polymerase chain reaction using starting materials which comprise a test sample and at least one set of genus-specific primers; and b.) detecting Haemobartonella species in the test sample in the event a Haemobartonella-sized amplicon is present. A method as described, wherein step b.) comprises gel electrophoresis is preferred, although any method for detecting amplicon(s) (e.g. size-differentiating chromatography) is within the scope of the present invention.
For instance, the above method can be used to identify both the specific presence, or the specific absence of a certain species of Haemobartonella. As an example, the present method could be used to test a sample using a primer set (one forward sequence, one reverse sequence, in amounts necessary to conduct PCR) designed to amplify, both H. felis Ohio and H. felis California, although the size of the amplicons would differ. In that instance, it is possible that the primers would amplify an amplicon unique for H. felis Ohio, and not H. felis California. The result would indicate the presence of H. felis Ohio as well as the absence of H. felis California. In fact, methods as described, wherein the primers are capable of amplifying uniquely-sized amplicons from H. felis Ohio and H. felis California is a preferred embodiment of the present invention. However, methods wherein the primers are capable of amplifying uniquely-sized amplicons for every Haemobartonella species are also preferred.
Moreover, the present invention is not limited to the use of only one set of genus-specific primers. The methods herein also include those wherein a second set of primers is used, for example, for nested PCR. However, methods wherein PCR is conducted using one set of genus-specific primers is preferred.
Methods which utilize primers designed using conserved sequences in or flanking the Haemobartonella 16S region are within the scope of the present invention. A preferred region for designing forward primers for the present invention is the region spanning nucleotides 175-425. Not all bases are identical in these regions, but those in the art are aware of primer design strategy in light of non-identical sequences. A preferred region for designing reverse primers for the present invention is the region spanning nucleotides 455-700. Not all bases are identical in these regions, but those in the art are aware of primer design strategy in light of non-identical sequences.
Methods as above wherein the genus-specific forward primer comprises a sequence selected from the group consisting of SEQ ID NO 1 and SEQ ID NO 3 are most preferred. Methods as described in the previous paragraph wherein the Haemobartonella genus-specific reverse primer comprises a sequence selected from the group consisting of SEQ ID NO 2 and SEQ ID NO 4 are most preferred.
Also provided in the present invention are methods to detect Haemobartonella-caused disease in a mammal, comprising: a.) conducting polymerase chain reaction using starting materials which comprise a test sample and at least one set of genus-specific primers; and b.) detecting Haemobartonella-caused disease in the test sample in the event a Haemobartonella-sized amplicon is present. A method as in this paragraph, wherein the Haemobartonella-caused disease is anemia is preferred.
Specifically the present invention also provides methods to detect anemia in a mammal, comprising: a.) conducting polymerase chain reaction using starting materials which comprise at least one set of genus-specific primers capable of amplifying H. felis Ohio and H. felis California nucleic acid, and a test sample; and b.) detecting feline infectious anemia in the test sample in the event a H. felis Ohio or a H. felis California-sized amplicon is present.
The genus-specific primers for the above assay can be designed using the H. felis Ohio (Genbank Accession Number 95297) sequence, designated herein as SEQ ID NO:5 and the H. felis California (Genbank Accession Number 88564) sequence, designated herein as SEQ ID NO:6.
The assays described herein comprise both a PCR step and an amplicon size-determination step. PCR can be conducted according to techniques known to one of skill in the art, including, for example, thermocycle PCR and isothermal PCR. A number of printed publications describe these procedures. For instance Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989), Ausubel et al., Current Protocols in Molecular Biology (Greene Publishing Associates, Inc., 1993) and Walker et al., 89 Proc Natl Acad Sci USA 392 (1992) describe typical parameters. Moreover, journal articles by investigators studying the organisms of interest will typically contain details about PCR amplification of the organisms"" nucleic acid.
For example, thermocycle PCR is conducted as follows: a sample is taken for amplification. Then, a thermocycler is used (at alternatingly high and low temperatures) to promote a cycle between a.) dissociation of double stranded nucleic acid; and b.) hybridization of the primers to any sample nucleic acid; and c.) subsequent synthesis of complementary nucleic acid. When the primers are bound to a nucleic acid in the test sample, the polymerase synthesizes a nucleic acid complementary to the sample nucleic acid, and when the primers are not bound, no synthesis takes place. A suitable biological sample includes, but is not limited to, a bodily fluid composition or a cellular composition. A bodily fluid refers to any fluid that can be collected (i.e., obtained) from an animal, examples of which include, but are not limited to, blood, serum, plasma, urine, tears, aqueous humor, cerebrospinal fluid (CSF), saliva, lymph, nasal secretions, milk and feces.
The second step in the described methods of the present invention is a size-determination of the PCR products generated. Size determination can be carried out according to any acceptable method, with gel electrophoresis being preferred. Methods for determining size of PCR products are described in Sambrook, supra and Ausubel, supra. Use of a control (identity known) sample or a sizing ladder is particularly helpful as well.
The primers of the present invention can be designed by aligning 16S regions from at least two Haemobartonella species and identifying primers which would amplify an amplicon having differences in absolute size as well as capable of priming polymerase chain reaction. Moreover, it is known in the art that primers are preferrably G-C rich, ideally more than 50% of the bases G or C. The length of the primer is usually chosen to minimize the chances of amplifying non-target nucleic acid, as well as minimize self-hybridization. Primers are typically 17 to 30 bases in length, although there are no absolute rules with regard to length or G-C content. For the purposes of the present invention, other parameters may take precedent over the length or constitution of the primers. Certain computer programs (such as MacVector) are helpful in primer design and PCR condition optimization.
The present invention includes kits useful for distinguishing between or among Haemobartonella species, comprising at least one set of genus-specific primers. The present kits preferably further comprise a gel material, such as, but not limited to, agarose or acrylamide.
Nucleic acid compounds are also provided by the present invention. Specifically, compositions of matter comprising a set of genus-specific primers as described herein are included in the present invention. A particular forward Haemobartonella genus-specific primers comprising a sequence selected from the group consisting of SEQ ID NO 1 and SEQ ID NO 3 are preferred. Particular reverse Haemobartonella genus-specific primers comprising a sequence selected from the group consisting of SEQ ID NO 2 and SEQ ID NO 4 are also preferred.
The sequences described in the sequence listing can be shortened from the 5xe2x80x2 end, provided that the resulting sequence does not result in loss of specificity when the shortened sequence is used as a primer. Those shortened primers are also useful as a part of a genus-specific primer set. For example, those primers wherein the 5xe2x80x2 terminus of SEQ ID NO 1 or SEQ ID NO 2 is shortened by 1-10 bases are also within the scope of the present invention. Primers wherein the 5xe2x80x2 terminus of SEQ ID NO 1 or SEQ ID NO 2 is shortened by 1-8 bases are preferred. SEQ ID NO 3 and SEQ ID NO 4 are most preferred. Any of these sequences can be used as primers in the methods described.